(a) Field of the Invention
The present invention relates to a recombinant vector for transcription of the Newcastle disease virus (NDV) genome, a strain of attenuated recombinant NDV with a surface antigen of pathogenic NDV prepared by the vector, a method of preparing a recombinant NDV having low pathogenicity and high protective efficiency against Newcastle disease (ND) using the vector, and a vaccine against ND containing the recombinant NDV.
(b) Description of the Related Art
Newcastle Disease (ND), which is known as one of the most important internationally known kinds of livestock disease, is an acute thermal respiratory disease, and is the first communicable disease by law in Korea. If an unimmunized fowl is infected, the mortality is 100%. Because the Newcastle disease virus (NDV) commonly exists in Korea, many difficulties are expected for eradicating the disease. Also, because various Newcastle disease viruses are widespread in Southeast Asia, China, and Taiwan, all of which actively trade with Korea, and because these viruses are potentially very dangerous factor, an urgent need to develop an Asia-type Newcastle disease vaccine exists.
The Newcastle disease virus (NDV) is a single-stranded RNA virus belonging to the Avulavirus genus. The envelope of the Newcastle disease virus includes the Haemagglutinin-Neuraminidase (HN) protein which makes the virus bind to a host, and the Fusion (F) protein which makes the envelope fuse with the host cell. F and HN proteins are glycoproteins and are distributed on the surface of the viral envelope.
The F protein belongs to the type I membrane glycoprotein group and forms a trimeric structure (trimer). The F protein is made as a non-active precursor form (F0), and is divided into the disulfide linked subunits F1 and F2 when the precursor F0 molecule passes through Golgi membranes. This process exposes a hydrophobic domain at the amino terminus of the F1 subunit, wherein the domain performs important role in the biological activity of mature protein. The hydrophobic domain, called a fusion peptide, is well conserved in the F protein of paramyxovirus, and is directly involved in membrane fusion. The F protein of paramyxovirus includes common structure characteristics such as heptad repeats, and two regions being capable of forming an alpha helix structure. The longest heptad repeat A of the repeats is adjacent to the N-terminal hydrophobic fusion peptide of F1, and heptad repeat B is close to the upper part of transmembrane region. Heptad repeat B consists of a series of well-conserved Leucine or Isoleusine at every 7 residues.
HN protein belongs to the type II membrane glycoprotein and forms a tetramer on the surface of the viral envelope, to penetrate into a cell membrane (Gorman et al., 1988; Ng et al., 1989). The HN protein causes virion to locate on the host cell surface via binding to sialic acids of glycoconjugates. The HN protein is divided into the three regions of a transmembrane domain, a stalk domain and a globular domain. Both a binding site of an antigenic receptor and an active site of neuraminidase locate on the globular domain. An active site of fusion induction locates on the stalk domain, and interacts with the F protein (Sergei et al., 1993). The expected structure of the stalk domain is an alpha-helix structure, with two heptad repeats including the heptad repeat A (at the 74th-88th positions) and the heptad repeat B (at the 96th-110th position). Also, it has been reported that any mutation breaking the structure reduces the receptor binding and the neuraminidase activity. Moreover, it has been reported that a mutation capable of destroying a structure causes decrease of the receptor binding and neuraminidase activities.
According to the level of disease in chicken, NDV is classified with the following pathogenic types (pathotypes): 1) viscerotropic velogenic (high-pathogenic) NDV showing digestive organ lesions and high mortality; and neurotropic velogenic NDV mainly showing respiratory and neurological symptom, and high mortality; 2) mesogenic NDV showing low mortality, but acute respiratory and neurological symptoms in some of the poultry; 3) lentogenic (low-pathogenic) and apathogenic NDV causing slight illness or asymptomatic respiratory infection.
In order for the NDV to infect a cell, it is necessary for the precursor glycoprotein F0 to be cleaved into F1 and F2. This post-translational cleavage is intervened by proteases of a host cell. If the cleavage does not occur, non-infectious virions are generated, and the virus replication cannot progress. The F0 protein of a virulent virus can be cleaved by various proteases, but the F0 protein of a low toxicity virus is restricted in terms of susceptibilities, and particularly the low toxicity virus is only capable of growing in a specific host cell type.
Whereas the lentogenic virus is only reproduced in a region that has trypsin-like enzymes, including respiratory organs or intestinal tract, because the virulent viruses are reproduced in various regions including tissues and organs, and therefore the virulent viruses cause a fetal systemic infection.
By the amino acid inspection of the Fo precursor, it is identified that the lentogenic viruses have a single arginine (R) connecting F2 and F1 subunit, whereas strains with toxicity of more than the mesogenic have additional basic amino acids forming two pairs such as K/R-X-K/R-R-F on the cleavage region. Moreover, the F2 chain of the strains with pathogenicity of more than the mesogenic is generally is disclosed by Phenylalanine residue, whereas the F2 chain of the strains with pathogenicity of less than the lentogenic is generally disclosed by Leucine.
In the U.S., a killed vaccine has been used for the identification of the Newcastle disease (Hofstad, 1953). By observation that a part of the enzootic disease virus only generates mild disease, for the first time, the mesogenic live vaccine Roakin was developed, and subsequently, milder Hitchner B1 and LaSota (Goldhaft, 1980) was developed.
One of the main advantages of live vaccines is the capability of administering by using a mass application method of a low-cost. A conventional application method is to administer the vaccine through drinking water.
The mass application of a live vaccine through spray and aerosols is very useful, because many birds can be more quickly administered with the vaccine and thereby vaccinated. It is important to control nozzles wherein particles are generated, to achieve an exacting particle size.
Recently used live vaccines have some problems. Because these vaccines still have a little pathogenicity, occasionally side effects of the vaccine can occur. Moreover, because antibodies inherited from the maternal line neutralize live vaccine viruses, successful immunity formation can be interfered with. Therefore, it is important to use an exceedingly mild virus, to perform the first vaccination, and a vaccine that is able to overcome the maternal antibody is required.
A killed vaccine is usually produced from infectious allantoic fluid mixed with appropriate supplements, and it is treated with formalin or beta-propiolactone, to kill the virus. The vaccine is administered to muscles or through subcutaneous injection, but it has a disadvantage due to a high-cost for its production and application.
Recently, the antigenicity of velogenic NDV generated both domestically and in foreign countries has been presumed to show many differences compared with the vaccine strain. For this reason, it can be concluded that the discovery of an outdoor strain with many differences with a genotype of the vaccine strain may occur, and if the vaccine antibody titer is not high enough, it is able to prevent mortality, but it is unable to prevent from decreasing in egg-laying rate, etc.
Depending on a phylogenic analysis based on the partial sequence of the F gene, the genotype of NDV is classified as genotype I to genotype IX. Molecular epidemiologically, most Newcastle disease viruses distributed in Korea belong to genotype VI and genotype VII. In the case of genotype VI, even though a variant strain may occur because of intensive vaccination, relatively lowly isolated than the genotype VII, and primarily only the genotype VII has been isolated after 2000, the possibility of its extermination is considered. Consequently, sequencing analysis, a determination of recent NDV gene sequences through genome project, and a molecular epidemiological research through gene comparison with world-wide NDV that is registered in the GenBank are very important for developing an optimal vaccine strain.
At present, conventionally used killed oil vaccine of Newcastle disease (ND) is produced by using lentogenic NDV such as Clone 30 or a LaSota strain, and the production of a killed vaccine using velogenic NDV is prohibited because of safety problems. Therefore, the necessity for technology for producing an ND vaccine that is safer, more economical, and in which the antigen is similar to the field strain is increased, and the development of a vaccine using reverse genetics technology is mostly closed technology to this demand.
The reverse genetics technology of a negative strand RNA virus is proposed as a technology for a rescue of infectious virus from the virus genome (U.S. Pat. No. 5,166,057). Even though this technology was originally proposed to manipulate the influenza virus genome, it can be successfully applied to various segment and non-segment negative strand RNA viruses, including Rabies virus, Respiratory Syncytial virus, and Sendai virus.
The present inventors developed a novel vaccine strain using the reverse genetics technology as described above, and as a result; the technology of producing safe ND vaccine strain with antigenicity similar to that of the field strain is developed.